The overall objectives of this research are to design, synthesize, and test highly specific active site inhibitors of glyoxalase. The types of substrate-related molecules to be considered will include mechanism-based suicide inactivators, enediol-proton transfer mechanism-based reversible inhibitors, and affinity label inactivators. These inhibitors, especially the irreversible ones that covalently bond to protein at the active site, will help establish the environment at this site. Because these inhibitors will be very selective for inhibiting glyoxalase I, all new inhibitors will be submitted to NCI for in vivo testing as effective antitumor agents. In addition, heavy atom kinetic isotope effects on the glyoxalase I reaction will be examined to explore the possibility of using such techniques as a means to differentiate 1,2-hydride shift mechanisms from enediol-proton transfers.